ABSTRACT
Figure 9.1 Production of amylose by combined use of sucrose or cellobiose phosphorylase and phosphorylase.
were higher than those from Glc-1-P. The molecular weight of amylose was strictly controlled by the sucrose/primer molar ratio. Furthermore, the Mw/Mn of the amylose of all sizes was close to 1, indicating that all the synthesized amylose had a narrow molecular weight distribution, which is same as that of amylose produced using Glc-1-P. The amyloses with molecular weights less than 71 × 103were produced as insoluble particles, while those with molecular weights more than 305 × 103 were produced in the solution. These results suggested that the properties of amylose differ according to the molecular weights. For the purpose of providing Glc-1-P, use of cellobiose phosphorylase combined with phosphorylase was also examined (Fig. 9.1). Cellobiose phosphorylase catalyzes a phosphorolytic reaction of cellobiose in the presence of Pi to produce Glc-1-P and glucose [8]. When partially purified cellobiose phosphorylase was incubated with cellobiose and phosphorylase in the presence of Pi, various sizes of amylose (from 4.2 × 104 to 7.3 × 105) were produced [6,7]. However, the yield (38.6%) was not as high as that in the aforementioned method using sucrose phosphorylase. In order to improve the yield of amylose, mutarotase and glucose oxidase were added to the initial reaction mixture [9]. These enzymes were expected to remove the glucose derived by the cellobiose-catalyzed reaction, and thus, shift the equilibrium state to phosphorolysis. The yield of amylose increased to 64.8% by the action of these enzymes. Cellulose is the most abundant biomass resource on the earth, and its effective use is an important research project, leading to the sustainable society in the future. On the basis of this viewpoint, the conversion of cellobiose into amylose is of great interest. 9.3 Synthesis of Branched Glucan by Combined
Use of Phosphorylase with Branching EnzymeBranching enzyme (BE, EC 2.4.1.18) catalyzes transfer of the glucan chain from one α-(14)-glucan molecule to a glucan acceptor to form a new α-(16)-linkage. The enzyme activity is widely distributed in bacteria, yeasts, plants, and animals, which is responsible for the formation of branching structure in amylopectin and glycogen. The BE genes from several thermophilic microorganisms, such
as B. stearothermphilus [10] and Aquifex aeolicus [11], have been isolated. The BE genes were expressed in various host strains, and the enzymes were well characterized. Branched glucan can be produced by the combined action of phosphorylase and BE on Glc-1-P in the presence of an adequate primer (Fig. 9.2) [12,13]. The molecular weight and branching pattern of the product are expected to be controlled by the Glc-1-P/primer ratio as in the case of amylose synthesis by the phosphorylase-catalyzed polymerization, and by the relative BE/phosphorylase activity ratio, respectively. Thus, various branched glucans were produced by using different BE/phosphorylase activity ratios. The branched glucans produced at high BE/phosphorylase activity ratios had more frequently branching points than those produced at low BE/phosphorylase ratios.
